This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. (1) DXV VP1: The viability of all viruses is dependent upon faithful replication of their entire genome, including terminal sequences. Many DNA and RNA viruses use protein primers, and produce genomic DNA/RNA molecules with the 5` end covalently linked to a polypeptide, often called VPg or terminal protein (TP). Birnaviruses form a unique family of dsRNA viruses with a VPg-linked genome. Here we propose to use birnaviruses as a paradigm to elucidate the structural basis of viral protein priming in genome replication. The drosophila X virus (DXV) in the birnavirus family encodes polypeptide VP1 that has both the polymerase and viral primer function. We have now grown DXV VP1 crystals that diffract to 3.5 angstrom at home. Our plan is to collect a high-resolution native data set at CHESS and also to prepare several heavy atom derivatives. Because a variety of viruses that cause human diseases (e.g., picornaviruses, caliciviruses, adenoviruses and heptadnaviruses) initiate genome replication by protein priming, the proposed research will provide promising leads for developing new antiviral compounds, as viral polymerases are often targets for antiviral therapy. (2) Influenza nucleoprotein: Influenza A viruses, which cause highly contagious, acute respiratory illnesses in humans, are a group of negative-strand (-) RNA viruses. Like other (-)RNA viruses, the genome of influenza A viruses, eight segments in total, is encapsidated in the form of ribonucleoprotein (RNP) complexes. The nucleoprotein (NP), the major protein component of RNPs, binds along the entire length of each genomic RNA segment, forming the double-helical RNP structure found in mature virus. The crystal structure of influenza A virus NP has recently been determined in our laboratory, and it shows an overall fold and topology vastly different from those of rhabdoviruses and borna disease virus, both members of Mononegavirale order. Based on calculated electrostatic potential, the RNA binding site appears to be located on the outer surface of NP oligomers, suggesting that viral RNA is likely to be exposed on the exterior of viral RNP complexes. In contrast, NP from rhabdoviruses binds to RNA at the interior of their oligomeric complexes. To determine how influenza virus NP binds RNA, we have now obtained several forms of crystals of NP bound to RNA. These crystals are thin-plates that are only 0.2mm wide. Our plan is to collect a high-resolution data for the complex and to solve the crystal structure using molecular replacement. (3) Penicillium stoloniferum Virus F (PsV-F): PsV-F is a double-stranded RNA virus in the Partitiviridae family. Previous studies on many dsRNA viruses suggest that these viruses contain an intact core capsid that protects the dsRNA genome and serves as the machinery for producing viral mRNAs during virus infection. In the past several years, X-ray crystal structures of members in the reovirus, birnavirus and totivirus family have been determined, and they show interesting similarities as well as distinct features in their viral capsid structure and function. As a dsRNA virus with only two dsRNA segments, it is important to determine whether PsV-F is similar capsid architecture as other dsRNA with multiple RNA segments. We have already obtained crystals of PsV-F that diffract to at least 3.6 angstrom at home. These crystals cannot be frozen, however. Our plan is to collect a complete data set at room temperature from capillary-mounted crystals. The crystal structure will be solved using EM reconstruction as molecular replacement models.